[3+2]-Cycloaddition of Nitrile Imines to Parabanic Acid Derivatives—An Approach to Novel Spiroimidazolidinediones

Approximately 1,3-Dipolar cycloaddition of imidazolidine derivatives containing exocyclic double bonds is a convenient method of creating spiro-conjugated molecules with promising anticancer activity. In this work, the derivatives of parabanic acid (2-thioxoimidazolidine-4,5-diones and 5-aryliminoimidazolidine-2,4-diones) were first investigated as dipolarophiles in the reactions with nitrile imines. The generation of nitrile imines was carried out either by the addition of tertiary amine to hydrazonoyl chlorides «drop by drop» or using the recently proposed diffusion mixing technique, which led to ~5–15% increases in target compound yields. It was found that the addition of nitrile imines to C=S or C=N exocyclic double bonds led to 1,2,4-thiazolines or triazolines and occurred regioselectively in accordance with the ratio of FMO coefficients of reactants. The yield of the resulting spiro-compound depended on the presence of alkyl substituents in the nitrile imine structure and was significantly decreased in reactions with imines with strong electron donor or electron-withdrawing groups. Some of the obtained compounds showed reasonable in vitro cytotoxicity. IC50 values were calculated for HCT116 (colon cancer) cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test.


Introduction
Nitrile imines are widely recognized for their high reactivity in 1,3-dipolar cycloaddition reactions, both in carbon-carbon multiple bonds and carbon-heteroatom bonds [1].The reactions of nitrile imines with asymmetric dipolarophiles usually proceed regio-and chemoselectively [2].The addition of nitrile imine to the C=C double bond results in the formation of a pyrazoline fragment, which is found in compounds possessing a broad spectrum of biological activities, such as anti-inflammatory [3], antiviral, antimicrobial [4], analgesic, immunosuppressive, antibacterial [5], anticancer [6], antidepressant, and neuroprotective [7] properties (Figure 1).The discovery of these properties has made the reactions of dipolar cycloaddition of nitrile imines a common tool in the synthesis of biologically active molecules and natural compounds [8].
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 2 of 26 depressant, and neuroprotective [7] properties (Figure 1).The discovery of these properties has made the reactions of dipolar cycloaddition of nitrile imines a common tool in the synthesis of biologically active molecules and natural compounds [8].Hydantoins exhibit a wide range of biological activity, among which antitumor, antibacterial, anticonvulsant, antiepileptic, and antiarrhythmic properties, as well as action as muscle relaxants, can be distinguished [9].The introduction of methylidene derivatives of hydantoins and thiohydantoins in the 1,3-dipolar cycloaddition reaction enables the preparation of spiro-conjugated compounds containing several heterocyclic pharmacophore fragments in one molecule [10][11][12] (Figure 2a,b).Sterically constrained structure of spiro-conjugated molecules coupled with bioactive fragments suggests the appearance of noticeable cytotoxic properties in the resulting compounds and thereby causes interest not only for organic chemistry but also for medical chemistry.Despite the fact that 5-arylidene(thio)hydantoins have long been known as dipolarophiles [12,13], their analogues containing exocyclic C=N bonds have not been previously investigated in such reactions.
Figure 2. A total of 1,3-dipolar cycloaddition reactions of nitrile imines to (a) 5-methylidene hydantoin [11,14]; (b) 5-arylidene thiohydantoins [10,13]; and (c) 5-arylimino hydantoins (this work).Hydantoins exhibit a wide range of biological activity, among which antitumor, antibacterial, anticonvulsant, antiepileptic, and antiarrhythmic properties, as well as action as muscle relaxants, can be distinguished [9].The introduction of methylidene derivatives of hydantoins and thiohydantoins in the 1,3-dipolar cycloaddition reaction enables the preparation of spiro-conjugated compounds containing several heterocyclic pharmacophore fragments in one molecule [10][11][12] (Figure 2a,b).Sterically constrained structure of spiro-conjugated molecules coupled with bioactive fragments suggests the appearance of noticeable cytotoxic properties in the resulting compounds and thereby causes interest not only for organic chemistry but also for medical chemistry.Despite the fact that 5arylidene(thio)hydantoins have long been known as dipolarophiles [12,13], their analogues containing exocyclic C=N bonds have not been previously investigated in such reactions.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 2 of 26 depressant, and neuroprotective [7] properties (Figure 1).The discovery of these properties has made the reactions of dipolar cycloaddition of nitrile imines a common tool in the synthesis of biologically active molecules and natural compounds [8].Hydantoins exhibit a wide range of biological activity, among which antitumor, antibacterial, anticonvulsant, antiepileptic, and antiarrhythmic properties, as well as action as muscle relaxants, can be distinguished [9].The introduction of methylidene derivatives of hydantoins and thiohydantoins in the 1,3-dipolar cycloaddition reaction enables the preparation of spiro-conjugated compounds containing several heterocyclic pharmacophore fragments in one molecule [10][11][12] (Figure 2a,b).Sterically constrained structure of spiro-conjugated molecules coupled with bioactive fragments suggests the appearance of noticeable cytotoxic properties in the resulting compounds and thereby causes interest not only for organic chemistry but also for medical chemistry.Despite the fact that 5-arylidene(thio)hydantoins have long been known as dipolarophiles [12,13], their analogues containing exocyclic C=N bonds have not been previously investigated in such reactions.
Unfortunately, we did not find any data in the literature on the biological properties of 5-arylidene(thio)hydantoins.However, the recent work of Ribeiro [15] revealed that the replacement of a pyrazoline ring by a triazoline ring in the spirooxindole core altered the antiproliferative profile.Spiropyrazolines showed in general less activity and selectivity towards tested cancer cell lines over the non-cancer-derived cell line than spirotriazolines with the same substitution pattern.Consequently, we assume that the products of the addition of nitrile imines to 5-imino hydantoins studied in this work should also have pronounced cytotoxicity.
There are in the literature a few examples where compounds with exocyclic C=N bonds were used as dipolarophiles.However, the available data demonstrated a good reactivity of the imino group in comparison with the trisubstituted bond in alkenes [16].In all cases, the addition of nitrile imines to the C=N bond occurs regioselectively with the formation of 1,2,4-triazole [1].
In a recent work by S.H. Ungoren [17], it has been shown that the interaction of N,N-disubstituted parabanic acids with aryliminophosphoranes leads to the formation of 5imino-substituted hydantoins with a good yield.Among the data available in the literature on the chemical properties of 5-substituted hydantoins in the 1,3-dipolar cycloaddition reactions, we found no examples of the interaction of some 1,3-dipoles and an exocyclic bond C=N of such compounds.
Therefore, in this paper, we investigated the reactivity of N-and N,N'-substituted thioparabanic acids in reactions of 1,3-dipolar cycloaddition.In addition, a one-pot method was proposed for the synthesis of 5-arylimino-1,3-diphenylimidazolidine-4-ones, which were further used as dipolarophiles in reactions with nitrile imines.The transformations discussed in this article are summed up in Scheme 1.Unfortunately, we did not find any data in the literature on the biological properties of 5-arylidene(thio)hydantoins.However, the recent work of Ribeiro [15] revealed that the replacement of a pyrazoline ring by a triazoline ring in the spirooxindole core altered the antiproliferative profile.Spiropyrazolines showed in general less activity and selectivity towards tested cancer cell lines over the non-cancer-derived cell line than spirotriazolines with the same substitution pattern.Consequently, we assume that the products of the addition of nitrile imines to 5-imino hydantoins studied in this work should also have pronounced cytotoxicity.
There are in the literature a few examples where compounds with exocyclic C=N bonds were used as dipolarophiles.However, the available data demonstrated a good reactivity of the imino group in comparison with the trisubstituted bond in alkenes [16].In all cases, the addition of nitrile imines to the C=N bond occurs regioselectively with the formation of 1,2,4-triazole [1].
In a recent work by S.H. Ungoren [17], it has been shown that the interaction of N,N-disubstituted parabanic acids with aryliminophosphoranes leads to the formation of 5-imino-substituted hydantoins with a good yield.Among the data available in the literature on the chemical properties of 5-substituted hydantoins in the 1,3-dipolar cycloaddition reactions, we found no examples of the interaction of some 1,3-dipoles and an exocyclic bond C=N of such compounds.
Therefore, in this paper, we investigated the reactivity of N-and N,N'-substituted thioparabanic acids in reactions of 1,3-dipolar cycloaddition.In addition, a one-pot method was proposed for the synthesis of 5-arylimino-1,3-diphenylimidazolidine-4-ones, which were further used as dipolarophiles in reactions with nitrile imines.The transformations discussed in this article are summed up in Scheme 1.
The preparation of substituted parabanic acids and their sulfur-containing analogues by the reaction of oxalyl chloride and corresponding thioureas was described more than a century ago [18], but for a long time, the structure of the resulting product remained the subject of discussion [18][19][20].The differences in the obtained results were caused by the possibility of the formation not only of substituted thioparabanic acid II but also of intermediate 2-iminothiazolidine-4,5-dione I, which in some cases could not be isolated (Scheme 2).At the same time, it was found that the isomerization of intermediate I into product II occurs quite easily, for example, when boiling in ethanol [20].

Synthesis of the Imidazolidine-4,5-Diones 1c-f
The preparation of substituted parabanic acids and their sulfur-contai logues by the reaction of oxalyl chloride and corresponding thioureas was more than a century ago [18], but for a long time, the structure of the resultin remained the subject of discussion [18][19][20].The differences in the obtained res caused by the possibility of the formation not only of substituted thioparaban but also of intermediate 2-iminothiazolidine-4,5-dione I, which in some cases be isolated (Scheme 2).At the same time, it was found that the isomerization o diate I into product II occurs quite easily, for example, when boiling in ethanol Scheme 2. Reaction of disubstituted thiourea and oxalyl chloride.
Investigating the reaction of diphenyl thiourea and oxalyl chloride, we f the formation of a particular of the two isomeric products can be conveniently m using NMR spectroscopy.Since 1,3-diphenyl-2-thioxoimidazolidine-4,5-dione plane of symmetry passing through the C=S bond and the middle of the C 4 -C 5 protons of aromatic fragments are equivalent and present in the spectrum as t signals.Approximately 3-Phenyl-2-(phenylimino)thiazolidine-4,5-dione 6 doe tain symmetry elements, so the signals of aromatic protons have a more comp plicity (Figure 3).In many publications, data on the formation of isomeric products I and II ( as a result of the reaction of diphenyl thiourea and oxalyl chloride were pres plicitly [17,21,22], and we could not find a clear methodology that allows us exclusively isomer II.By conducting reactions of various thioureas with oxaly we found that a thiazolidine-type product (I) was formed very easily.The pro obtained with a high yield after 1-2 h of boiling the reaction mixture in an apro with a low boiling point, such as dichloromethane.When boiling the reaction m Scheme 2. Reaction of disubstituted thiourea and oxalyl chloride.
Investigating the reaction of diphenyl thiourea and oxalyl chloride, we found that the formation of a particular of the two isomeric products can be conveniently monitored using NMR spectroscopy.Since 1,3-diphenyl-2-thioxoimidazolidine-4,5-dione 1c has a plane of symmetry passing through the C=S bond and the middle of the C 4 -C 5 bond, the protons of aromatic fragments are equivalent and present in the spectrum as two sets of signals.Approximately 3-Phenyl-2-(phenylimino)thiazolidine-4,5-dione 6 does not contain symmetry elements, so the signals of aromatic protons have a more complex multiplicity (Figure 3).In many publications, data on the formation of isomeric products I and II (Scheme 2) as a result of the reaction of diphenyl thiourea and oxalyl chloride were presented implicitly [17,21,22], and we could not find a clear methodology that allows us to obtain exclusively isomer II.By conducting reactions of various thioureas with oxalyl chloride, we found that a thiazolidine-type product (I) was formed very easily.The product was obtained with a high yield after 1-2 h of boiling the reaction mixture in an aprotic solvent with a low boiling point, such as dichloromethane.When boiling the reaction mixture in a more highly boiling solvent, such as acetonitrile, a noticeable amount of isomerized imidazolidine-type product (II) was formed in a few hours (about 10% after 2 h).We also found that chromatographic separation of isomers 6 and 1c is not feasible since 2-iminothiazolidine-4,5-dione 6 was completely transformed into 2-thioxoimidazolidine-4,5-dione 1c passing through silica gel.
In contrast to the spectrally distinguishable compounds 1c and 6, the determination of the structure of products obtained from oxalyl chloride and non-symmetric thioureas (R 1 ̸ = R 2 ) is a more difficult task since the result of the reaction can be both an imidazolidine product II and two isomeric thiazolidines I.In this regard, imidazolidine-2,4,5-triones 1a-b and 2-thioxoimidazolidine-4,5-diones 1c-f were obtained according to two different synthetic strategies.The first ones were obtained by boiling a mixture of oxalyl chloride and urea in DCM, followed by treatment with a solution of sodium bicarbonate to neutralize oxalyl chloride and hydrochloric acid residues.To obtain sulfur-containing products, we used acetonitrile as a solvent and flash chromatography as a purification method, after which only imidazolidine-type products 1c-f were isolated.The yields of all compounds 1a-f were good to excellent, with the exception of product 1f, which contains an unsubstituted N 3 atom (Table 1).In contrast to the spectrally distinguishable compounds 1c and 6, the determi of the structure of products obtained from oxalyl chloride and non-symmetric thi (R1 ≠ R2) is a more difficult task since the result of the reaction can be both an imid dine product II and two isomeric thiazolidines I.In this regard, imid dine-2,4,5-triones 1a-b and 2-thioxoimidazolidine-4,5-diones 1c-f were obtain cording to two different synthetic strategies.The first ones were obtained by bo mixture of oxalyl chloride and urea in DCM, followed by treatment with a solut sodium bicarbonate to neutralize oxalyl chloride and hydrochloric acid residues.tain sulfur-containing products, we used acetonitrile as a solvent and flash chrom raphy as a purification method, after which only imidazolidine-type products 1cisolated.The yields of all compounds 1a-f were good to excellent, with the excep product 1f, which contains an unsubstituted N 3 atom (Table 1).

Synthesis of the 5-Aryliminoimidazolidine-2,4-Diones 2a-l
In a recent paper by Ş.H. Ungoren and colleagues [17], it was reported that tuted parabanic acids react with phosphonium ylides to form 5-alkyliden 5-imino-substituted hydantoins.We modified this technique and carried out the o aza-Wittig reaction without the isolation of intermediate aryliminophosphoranes.cally, a mixture of arylazide and triphenylphosphine in dry xylene was stirred at perature of about 50 °C until nitrogen release stopped (0.5 to 2 h).Imidazoldione added to the phosphorus ylide formed in situ, after which the mixture was boiled f The product was isolated using column chromatography and, if necessary, additi recrystallized from isopropanol.In all cases, imines 2a-j were obtained with good (Table 2).The resulting productʹs C=N double bond configuration was attributed cordance with the literature data on the structure of similar compounds [17].

Synthesis of the 5-Aryliminoimidazolidine-2,4-Diones 2a-l
In a recent paper by Ş.H. Ungoren and colleagues [17], it was reported that substituted parabanic acids react with phosphonium ylides to form 5-alkylidene and 5-iminosubstituted hydantoins.We modified this technique and carried out the one-pot aza-Wittig reaction without the isolation of intermediate aryliminophosphoranes.Typically, a mixture of arylazide and triphenylphosphine in dry xylene was stirred at a temperature of about 50 • C until nitrogen release stopped (0.5 to 2 h).Imidazoldione 1 was added to the phosphorus ylide formed in situ, after which the mixture was boiled for 2 h.The product was isolated using column chromatography and, if necessary, additionally recrystallized from isopropanol.In all cases, imines 2a-j were obtained with good yields (Table 2).The resulting product's C=N double bond configuration was attributed in accordance with the literature data on the structure of similar compounds [17].perature of about 50 °C until nitrogen release stopped (0.5 to 2 h).Imidazoldione added to the phosphorus ylide formed in situ, after which the mixture was boiled f The product was isolated using column chromatography and, if necessary, additi recrystallized from isopropanol.In all cases, imines 2a-j were obtained with good (Table 2).The resulting productʹs C=N double bond configuration was attributed cordance with the literature data on the structure of similar compounds [17].As previously described [22], Wittig reactions with thiazolidine 6 give the corresponding trisubstituted alkene.Therefore, carrying out a similar aza-reaction to obtain thiazolidine with two C=N bonds simultaneously at positions C 2 and C 5 seemed quite real.Contrary to this, we unexpectedly found that compound 6 in the reactions with phosphonium ylides, formed in situ from PPh 3 and corresponding arylazides, undergoes a one-pot aza-Wittig reaction to furnish the same product as it was formed at the identical conditions from thioparabanic acid 1c, but with a significantly lower yield (Scheme 3).The presence of an exocyclic C=S bond in compounds 2i and 2j was indicated by the appearance of a carbon atom in the thiocarbonyl group signal at ~180 ppm in the 13 C NMR spectrum.The occurrence of this signal in the crude reaction mixture spectrum suggests isomerization of the thiazolidine cycle into imidazolidine was initiated during the reaction and did not occur during the chromatographic purification of imines 2i and 2j.As previously described [22], Wittig reactions with thiazolidine 6 give the corresponding trisubstituted alkene.Therefore, carrying out a similar aza-reaction to obtain thiazolidine with two C=N bonds simultaneously at positions C 2 and C 5 seemed quite real.Contrary to this, we unexpectedly found that compound 6 in the reactions with phosphonium ylides, formed in situ from PPh3 and corresponding arylazides, undergoes a one-pot aza-Wittig reaction to furnish the same product as it was formed at the identical conditions from thioparabanic acid 1c, but with a significantly lower yield (Scheme 3).The presence of an exocyclic C=S bond in compounds 2i and 2j was indicated by the appearance of a carbon atom in the thiocarbonyl group signal at ~180 ppm in the 13 C NMR spectrum.The occurrence of this signal in the crude reaction mixture spectrum suggests isomerization of the thiazolidine cycle into imidazolidine was initiated during the reaction and did not occur during the chromatographic purification of imines 2i and 2j.Scheme 3. Aza-Wittig reaction of isomers 1c and 6.

1,3-Dipolar Cycloaddition of Nitrile Imines to 2-Thioxoimidazolidine-4,5-Diones 1c-f and 3-Phenyl-2-(Phenylimino)Thiazolidine-4,5-Dione 6
Despite the widespread recognition of 1,3-dipolar cycloaddition of thiohydantoins and their 5-methylidene/arylidene substituted analogues for creating spiro-linked compounds with 1,3,4-thiadiazoline [10,11,13] fragments, there are no examples in the literature of 5-oxothiohydantoins (thioparabanic acids) being used as dipolarophiles.Nitrile imines are highly active 1,3-dipoles, so they are usually generated in situ from the corresponding precursors.To obtain nitrile imines, we used hydrazonoyl chlorides 3a-g (Figure 4), which are readily available by the method [11].Despite the widespread recognition of 1,3-dipolar cycloaddition of thiohydantoins and their 5-methylidene/arylidene substituted analogues for creating spiro-linked compounds with 1,3,4-thiadiazoline [10,11,13] fragments, there are no examples in the literature of 5-oxothiohydantoins (thioparabanic acids) being used as dipolarophiles.Nitrile imines are highly active 1,3-dipoles, so they are usually generated in situ from the corresponding Our laboratory has recently developed the so-called "diffusion reagent mixing technique", which allows the gradual generation of 1,3-dipoles in solution and effectively suppresses the processes of their unwanted dimerization [14].In many cases, the diffusion mixing technique makes it possible to significantly increase the yield of the target [3+2]-cycloaddition products and is especially effective in cases of interaction with highly reactive dipoles.For the reactions of 2-thioxoimidazolidine-4,5-diones 1c-f with nitrile imines, we compared two synthetic approaches, which differed in the way of introducing in the reaction mixture a base generating a dipole from hydrazonoyl chloride 3a-d.In the first "classical" method, the base solution was added drop by drop to a reaction mixture containing a dipolarophile 1c-f and a dipole precursor 3a-d.In the second approach, we used the equipment for the diffusion reagent mixture (see [15] and Supplementary Information), whereby the volatile base spontaneously diffused into a solution of reagents.
Both methods of reagent mixing enabled us to obtain spiro compounds 4a-g with good yields (Table 3).It was found that the use of the diffusion mixing technique resulted in target product yields comparable to or exceeding the results obtained by adding amine drop by drop.Compound 4c was obtained with a yield near quantitative using both methods.The only Our laboratory has recently developed the so-called "diffusion reagent mixing technique", which allows the gradual generation of 1,3-dipoles in solution and effectively suppresses the processes of their unwanted dimerization [14].In many cases, the diffusion mixing technique makes it possible to significantly increase the yield of the target [3+2]-cycloaddition products and is especially effective in cases of interaction with highly reactive dipoles.For the reactions of 2-thioxoimidazolidine-4,5-diones 1c-f with nitrile imines, we compared two synthetic approaches, which differed in the way of introducing in the reaction mixture a base generating a dipole from hydrazonoyl chloride 3a-d.In the first "classical" method, the base solution was added drop by drop to a reaction mixture containing a dipolarophile 1c-f and a dipole precursor 3a-d.In the second approach, we used the equipment for the diffusion reagent mixture (see [15] and Supplementary Information), whereby the volatile base spontaneously diffused into a solution of reagents.
Both methods of reagent mixing enabled us to obtain spiro compounds 4a-g with good yields (Table 3).Our laboratory has recently developed the so-called "diffusion reagent mixing technique", which allows the gradual generation of 1,3-dipoles in solution and effectively suppresses the processes of their unwanted dimerization [14].In many cases, the diffusion mixing technique makes it possible to significantly increase the yield of the target [3+2]-cycloaddition products and is especially effective in cases of interaction with highly reactive dipoles.For the reactions of 2-thioxoimidazolidine-4,5-diones 1c-f with nitrile imines, we compared two synthetic approaches, which differed in the way of introducing in the reaction mixture a base generating a dipole from hydrazonoyl chloride 3a-d.In the first "classical" method, the base solution was added drop by drop to a reaction mixture containing a dipolarophile 1c-f and a dipole precursor 3a-d.In the second approach, we used the equipment for the diffusion reagent mixture (see [15] and Supplementary Information), whereby the volatile base spontaneously diffused into a solution of reagents.
Both methods of reagent mixing enabled us to obtain spiro compounds 4a-g with good yields (Table 3).It was found that the use of the diffusion mixing technique resulted in target product yields comparable to or exceeding the results obtained by adding amine drop by drop.Compound 4c was obtained with a yield near quantitative using both methods.The only It was found that the use of the diffusion mixing technique resulted in target product yields comparable to or exceeding the results obtained by adding amine drop by drop.Compound 4c was obtained with a yield near quantitative using both methods.The only exception was compound 4b, whose yield when using the diffusion mixing technique was significantly lower due to the formation of a by-product 7 (Scheme 4).
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 8 of 26 exception was compound 4b, whose yield when using the diffusion mixing technique was significantly lower due to the formation of a by-product 7 (Scheme 4).It is known that in several cases [11,23], the interaction of nitrile imines and cyclic dipolarophiles led to the destruction of the initial imidazolidine cycle instead of the creation of a spiro-jointed product.Probably, in our case, as in the above publications, the presence of a strong electron acceptor (nitro group) in the dipole structure stabilizes the zwitterionic intermediate III, leading to the formation of the product 7 (Scheme 4).At the same time, the fragmentation of the molecule apparently occurs only when two aromatic fragments are present in the structure of the thioparabanic acid (no formation of a by-product was observed when products 4f and 4g were obtained).An analogous influence of the nature of substituents on the tendency to fragmentation was previously observed in the formation of spiro compounds from 5-arylidene thiohydantoins and thiosemicarbazides [24].The structure of product 7 was proposed based on a match between the spectral data obtained by us and those described in the literature [25].
Optimizing the conditions for the reaction of nitrile imines and 2-thioxoimidazolidine-4,5-diones, we found out that the yield of the target spiro-conjugated product 4a-g for most substrates was primarily influenced by the electronic properties of the substituents and did not depend much on the solvent selection.The situation dramatically changed when product 7 with a disrupted imidazolidine fragment could be formed as a result of the reaction (Table 4).TLC analysis of the reaction mixture revealed that compound 7 in high-polar solvents was either formed in trace amounts (Entry 2) or was absent from the mixture (Entry 1).Furthermore, the use of slightly less polar acetone or chloroform in the reaction led to the formation of a large amount of by-product 7, in some cases even exceeding the amount of the target spiro compound 4b (Entry 4 and 5).The yield of product 4b slightly increased when the reaction mixture cooled (Entry 3), but the significance of this factor was negligible.It is known that in several cases [11,23], the interaction of nitrile imines and cyclic dipolarophiles led to the destruction of the initial imidazolidine cycle instead of the creation of a spiro-jointed product.Probably, in our case, as in the above publications, the presence of a strong electron acceptor (nitro group) in the dipole structure stabilizes the zwitterionic intermediate III, leading to the formation of the product 7 (Scheme 4).At the same time, the fragmentation of the molecule apparently occurs only when two aromatic fragments are present in the structure of the thioparabanic acid (no formation of a by-product was observed when products 4f and 4g were obtained).An analogous influence of the nature of substituents on the tendency to fragmentation was previously observed in the formation of spiro compounds from 5-arylidene thiohydantoins and thiosemicarbazides [24].The structure of product 7 was proposed based on a match between the spectral data obtained by us and those described in the literature [25].
Optimizing the conditions for the reaction of nitrile imines and 2-thioxoimidazolidine-4,5-diones, we found out that the yield of the target spiro-conjugated product 4a-g for most substrates was primarily influenced by the electronic properties of the substituents and did not depend much on the solvent selection.The situation dramatically changed when product 7 with a disrupted imidazolidine fragment could be formed as a result of the reaction (Table 4).TLC analysis of the reaction mixture revealed that compound 7 in high-polar solvents was either formed in trace amounts (Entry 2) or was absent from the mixture (Entry 1).Furthermore, the use of slightly less polar acetone or chloroform in the reaction led to the formation of a large amount of by-product 7, in some cases even exceeding the amount of the target spiro compound 4b (Entry 4 and 5).The yield of product 4b slightly increased when the reaction mixture cooled (Entry 3), but the significance of this factor was negligible.The monosubstituted double bond -CH=CH 2 is known to be capable of reacting easily in the 1,3-dipolar cycloaddition, but in most cases, the thiocarbonyl group is a significantly more active dipolarophile than the carbon-carbon double bond [8].The difference in the reactivity of C=S and C=C groups made it possible to synthesize the compound 4g, the reaction product of the thioparabanic acid 1f containing an allyl substituent at the nitrogen atom.By employing the diffusion mixing technique, we managed to obtain the product 4g in a good yield (Table 3), while carrying out this reaction by adding triethylamine drop by drop led to the formation of a mixture of compounds that could not be qualitatively separated by chromatography.Based on the analysis of the spectral data obtained for this mixture, we assumed that the dropwise addition of a very dilute triethylamine solution to 1f and hydrazonoyl chloride 3b led to a local overconcentration of the generated nitrile imine, which was sufficient to attach the second dipole molecule to the already formed spiro compound 4g (Scheme 5).Presumably, the addition of nitrile imine to the carbon-carbon double bonds of the allyl substituent of the product 4g occurred analogously to reactions with monosubstituted olefins [1] and led to the formation of two regioisomers V and VI very similar in their chromatographic properties.In the case of diffusion, the unstable 1,3-dipole was present in the solution at a low concentration sufficient for reaction with only one most active C=X bond.In our study, when compound 6 was introduced into the reaction with nitrile imines, we established that the sulfur atomʹs tendency to isomerize to an exocyclic position prevailed over the reactivity of the 2-arylimino fragment.The presence of a second carbonyl group in the structure of compound 6 facilitated a process similar to that occurring during isomerization in 1c, which was described in [20].It appears that, unlike the process described in the article [27], interaction with the dipole leads to a cleavage of the S-C 5 bond instead of the S-C 2 bond (Scheme 7).In our study, when compound 6 was introduced into the reaction with nitrile imines, we established that the sulfur atomʹs tendency to isomerize to an exocyclic position prevailed over the reactivity of the 2-arylimino fragment.The presence of a second carbonyl group in the structure of compound 6 facilitated a process similar to that occurring during isomerization in 1c, which was described in [20].It appears that, unlike the process described in the article [27], interaction with the dipole leads to a cleavage of the S-C 5 bond instead of the S-C 2 bond (Scheme 7).Based on the obtained results, it can be concluded that 2-thioxoimidazolidine-4,5diones 1c-f possess the capability to undergo the 1,3-dipolar cycloaddition with nitrile imines obtained in situ from the corresponding hydrazonoyl chlorides 3a-d.As a result, heterocycles 4a-g consisting of spiro-conjugated imidazolidine and 1,2,4-thiadiazoline fragments were formed.The nitrile imine generation can be accomplished through both the "classical" method of adding the base drop by drop and the previously proposed diffusion mixing technique.The use of the latter enabled the site-selective attachment of the dipole to the C=S bond of 2-thioxoimidazolidine-4,5-dione 1f in the presence of a second dipolarophilic double bond in the molecule and the formation of compound 4g.The formation of imidazolidine cycle fragmentation product 7 was observed when spiro compounds were obtained from 1,3-diphenyl-2-thioxoimidazolidine-4,5-dione 1c and nitrile imine generated by the diffusion mixing technique.Cycloaddition of nitrile imines to 3-phenyl-2-(phenylimino)thiazolidine-4,5-dione 6 resulted in the same products 4a-d as were obtained from 1,3-diphenyl-2-thioxoimidazolidine-4,5-dione 1c.Approximately 5-Arylimino-1,3-diphenylimidazolidine-2,4,5-triones 2b-g were introduced into reactions with nitrile imines obtained in situ from hydrazonoyl halides 3b-g (Figure 4).Two alternative techniques were also used to generate dipoles: diffusion mixing and triethylamine dropwise addition (see detailed description in Section 2.3).Spiro compounds 5a-l were obtained in a moderate-to-high yield, which, however, was lower on average compared to those obtained for compounds 4 (Table 5).were obtained from 1,3-diphenyl-2-thioxoimidazolidine-4,5-dione 1c.
The diffusion mixing technique revealed a higher yield of the spiro compounds than when triethylamine was added drop by drop, except for products 5f and 5j obtained from imine 2e (R1 = 4-Me).The yield of the cycloaddition product was highly influenced by the electronic properties of the substituents in the initial imine; the presence of a strong donor or acceptor substituent at the R1 position led to a sharp decrease in the yield of the compounds 5b, 5g-i.The obtained results indicate that the C=N group of 5-arylimino-1,3-diphenylimidazolidine-2,4-diones is, in most cases, less active than the 2-thiocarbonyl group of compounds 1.When imines 2h-j containing a couple of reactive C=S and C=N bonds were introduced into the reactions with hydrazonoyl chlorides, we observed the formation of a complex mixture of products, presumably consisting of spiro compounds obtained as a result of the addition of a dipole along one of the C=S or C=N bonds, as well as the addition products of two dipole molecules along both of these bonds with two possible diastereomers formation.The diffusion mixing technique revealed a higher yield of the spiro compounds than when triethylamine was added drop by drop, except for products 5f and 5j obtained from imine 2e (R 1 = 4-Me).The yield of the cycloaddition product was highly influenced by the electronic properties of the substituents in the initial imine; the presence of a strong donor or acceptor substituent at the R 1 position led to a sharp decrease in the yield of the compounds 5b, 5g-i.The obtained results indicate that the C=N group of 5-arylimino-1,3-diphenylimidazolidine-2,4-diones is, in most cases, less active than the 2-thiocarbonyl group of compounds 1.When imines 2h-j containing a couple of reactive C=S and C=N bonds were introduced into the reactions with hydrazonoyl chlorides, we observed the formation of a complex mixture of products, presumably consisting of spiro compounds obtained as a result of the addition of a dipole along one of the C=S or C=N bonds, as well as the addition products of two dipole molecules along both of these bonds with two possible diastereomers formation.
We also tried to introduce 1,3-dipoles generated from hydrazonoyl chlorides 3 with N-heterocyclic substituents (see Table 5, R 2 = 2-pyridyl-or 5-(H-methyl-pyrazolinyl)) into reactions with imidazolidine-diones.However, the test reactions showed that under conditions optimized for aryl-substituted reagents, not only is it not possible to carry out prepatively the cycloaddition reactions of the corresponding nitrilylides with the formation of the target spiro-compounds, but even the starting hydrazonoyl chlorides 3 were obtained in low yields.It is possible that it occurs due to the presence of basic nitrogen atoms in the nitrogen-containing heterocycles, which can be protonated by HCl released during the dehydrohalogenation of a starting hydrazonyl halide.The reaction conditions when using heterocyclic derivatives, therefore, should be additionally optimized and will be the subject of our subsequent studies.
Considering the cycloaddition reactions of nitrile imines from the point of frontier molecular orbital (FMO) theory, they are referred to as dipoles, reactions with which can be controlled both by the interaction between the highest occupied molecular orbital of the dipole (HOMO dipole ) and the lowest unoccupied molecular orbital of the dipolarophile (LUMO dipolarophile ) and by the interaction between the LUMO of the dipole and the HOMO of the dipolarophile [28].Despite the limited quantity of calculated data regarding the reactions of these dipoles with imines, it is widely acknowledged that the regioselectivity of the 1,3-dipolar cycloaddition is governed by the relative disposition of dipole and dipolarophile frontier orbitals [28].Both with the control of the HOMO imine -LUMO dipole and with the overlap of the LUMO dipole -HOMO imine , the reaction leads to the formation of a 1,3,4triazole fragment [29].In all the cases examined, we observed a regioselective addition of nitrile imines to the C=N double bond of 5-arylimino-1,3-diphenylimidazolidine-2,4-diones, which corresponds to the theoretical principle regarding the course of such reactions.The structures of compounds 5c, 5g, and 5i were confirmed by X-ray diffraction analysis for the single crystals (Figure 5).We also tried to introduce 1,3-dipoles generated from hydrazonoyl chlorides 3 with N-heterocyclic substituents (see Table 5, R2 = 2-pyridyl-or 5-(H-methyl-pyrazolinyl)) into reactions with imidazolidine-diones.However, the test reactions showed that under conditions optimized for aryl-substituted reagents, not only is it not possible to carry out prepatively the cycloaddition reactions of the corresponding nitrilylides with the formation of the target spiro-compounds, but even the starting hydrazonoyl chlorides 3 were obtained in low yields.It is possible that it occurs due to the presence of basic nitrogen atoms in the nitrogen-containing heterocycles, which can be protonated by HCl released during the dehydrohalogenation of a starting hydrazonyl halide.The reaction conditions when using heterocyclic derivatives, therefore, should be additionally optimized and will be the subject of our subsequent studies.
Considering the cycloaddition reactions of nitrile imines from the point of frontier molecular orbital (FMO) theory, they are referred to as dipoles, reactions with which can be controlled both by the interaction between the highest occupied molecular orbital of the dipole (HOMOdipole) and the lowest unoccupied molecular orbital of the dipolarophile (LUMOdipolarophile) and by the interaction between the LUMO of the dipole and the HOMO of the dipolarophile [28].Despite the limited quantity of calculated data regarding the reactions of these dipoles with imines, it is widely acknowledged that the regioselectivity of the 1,3-dipolar cycloaddition is governed by the relative disposition of dipole and dipolarophile frontier orbitals [28].Both with the control of the HOMOimine-LUMOdipole gap and with the overlap of the LUMOdipole-HOMOimine, the reaction leads to the formation of a 1,3,4-triazole fragment [29].In all the cases examined, we observed a regioselective addition of nitrile imines to the C=N double bond of 5-arylimino-1,3-diphenylimidazolidine-2,4-diones, which corresponds to the theoretical principle regarding the course of such reactions.The structures of compounds 5c, 5g, and 5i were confirmed by X-ray diffraction analysis for the single crystals (Figure 5).The presence of substituents with pronounced electron-donating or electron-acceptor effects in the dipolarophile structure changes the energy of the frontier molecular orbitals, raising it (if the substituent has donor properties) or decreasing it (acceptor).As a result, one of the ways of overlapping the dipole and the dipolarophile frontier orbitals becomes more preferable [28].Upon analyzing the correlation between the yield of the resulting spiro compounds and the electronic properties of substituents in aromatic fragments, we found a number of patterns:

•
The presence of substituents with strong mesomeric effects in the aromatic ring of imine 2 decreased the yield of the spiro compound, regardless of whether the sub- The presence of substituents with pronounced electron-donating or electron-acceptor effects in the dipolarophile structure changes the energy of the frontier molecular orbitals, raising it (if the substituent has donor properties) or decreasing it (acceptor).As a result, one of the ways of overlapping the dipole and the dipolarophile frontier orbitals becomes more preferable [28].Upon analyzing the correlation between the yield of the resulting spiro compounds and the electronic properties of substituents in aromatic fragments, we found a number of patterns: • The presence of substituents with strong mesomeric effects in the aromatic ring of imine 2 decreased the yield of the spiro compound, regardless of whether the substituent was a donor or acceptor.For example, the yield decreased in the sequence 5c (R 1 = 4-Br) > 5g (R 1 = 4-OMe) > 5b (R 1 = 4-NO 2 ) (Table 5).
The side processes of the dipole dimerization, which are typical for the 1,3-dipolar cycloaddition, were suppressed during the diffusion addition of amine by generating a dipole at low concentration and with the addition of a base dropwise due to adding its dilute solution.Therefore, the decrease in the yields of target spiro compounds 5b and 5g-i (Table 5) is presumably attributed to the low imine reactivity instead of other factors impeding the dipole addition.In these cases, the conversion of the initial imines remained low even after several days of conducting the reaction, while the yield of the product changed insignificantly with an increase in the amount of hydrazonoyl chlorides or triethylamine added to the reaction.
It also should be noted that when the reacting nitrile imines were stabilized by the single aromatic substituent (compounds 4d in Table 4, 5k, and 5l in Table 5), the difference between the yields of the spiro compound 5 was more significant using different dipole generating techniques than for compounds 4, where the cycloaddition of nitrile imine occurred to the C=S bond.For instance, the difference in yields of the compound 5k obtained by diffusion mixing and by triethylamine dropwise addition was nearly twice as large as for the product 4d and reached almost 30%.This disparity may be caused by the facile dimerization of the dipole obtained from the hydrazonoyl halide 3d, with a relatively low reactivity of the dipolarophile.For compound 5l, the difference in yields of target spiro-compounds at different methods of dipole generation was not so significant (16%), apparently due to the greater activity of the C=N bond.
The surprising result was that substituents in the aromatic fragment of imine 2 did not have a significant steric effect on the yields of 5c (R 1 = 4-Br), 5d (R 1 = 2-Br), and 5e (R 1 = 2-Cl), which were comparable for both methods of dipole generation (dropwise or diffusion mixing).Despite this, the NMR spectra of the isolated products 5d and 5e demonstrated two sets of signals, indicating the formation of two isomers.These isomers in the case of compound 5d were separated by column chromatography (see Section 3.5), in contrast to the isomers of 5e, distinguishable by TLC analysis but hardly separable preparatively.It was also demonstrated using NMR 1 H that in the solution, individual isomers of compound 5d were gradually transformed into an isomeric mixture.In about a month, they reached an equilibrium ratio that coincided with that that had been previously determined for the reaction mixture.If necessary, this isomer mixture may be repeatedly separated.Based on [30], the observed phenomena can be explained by the atropoisomerism conferred by the repulsive interactions of ortho-substituent R 1 (the halogen atom) and the aromatic substituent R 2 at the neighboring nitrogen atom of the triazoline cycle (Figure 6).stituent was a donor or acceptor.For example, the yield decreased in the sequence 5c (R1 = 4-Br) > 5g (R1 = 4-OMe) > 5b (R1 = 4-NO2) (Table 5).
The side processes of the dipole dimerization, which are typical for the 1,3-dipolar cycloaddition, were suppressed during the diffusion addition of amine by generating a dipole at low concentration and with the addition of a base dropwise due to adding its dilute solution.Therefore, the decrease in the yields of target spiro compounds 5b and 5g-i (Table 5) is presumably attributed to the low imine reactivity instead of other factors impeding the dipole addition.In these cases, the conversion of the initial imines remained low even after several days of conducting the reaction, while the yield of the product changed insignificantly with an increase in the amount of hydrazonoyl chlorides or triethylamine added to the reaction.
It also should be noted that when the reacting nitrile imines were stabilized by the single aromatic substituent (compounds 4d in Table 4, 5k, and 5l in Table 5), the difference between the yields of the spiro compound 5 was more significant using different dipole generating techniques than for compounds 4, where the cycloaddition of nitrile imine occurred to the C=S bond.For instance, the difference in yields of the compound 5k obtained by diffusion mixing and by triethylamine dropwise addition was nearly twice as large as for the product 4d and reached almost 30%.This disparity may be caused by the facile dimerization of the dipole obtained from the hydrazonoyl halide 3d, with a relatively low reactivity of the dipolarophile.For compound 5l, the difference in yields of target spiro-compounds at different methods of dipole generation was not so significant (16%), apparently due to the greater activity of the C=N bond.
The surprising result was that substituents in the aromatic fragment of imine 2 did not have a significant steric effect on the yields of 5c (R1 = 4-Br), 5d (R1 = 2-Br), and 5e (R1 = 2-Cl), which were comparable for both methods of dipole generation (dropwise or diffusion mixing).Despite this, the NMR spectra of the isolated products 5d and 5e demonstrated two sets of signals, indicating the formation of two isomers.These isomers in the case of compound 5d were separated by column chromatography (see Section 3.5), in contrast to the isomers of 5e, distinguishable by TLC analysis but hardly separable preparatively.It was also demonstrated using NMR 1 H that in the solution, individual isomers of compound 5d were gradually transformed into an isomeric mixture.In about a month, they reached an equilibrium ratio that coincided with that that had been previously determined for the reaction mixture.If necessary, this isomer mixture may be repeatedly separated.Based on [30], the observed phenomena can be explained by the atropoisomerism conferred by the repulsive interactions of ortho-substituent R1 (the halogen atom) and the aromatic substituent R2 at the neighboring nitrogen atom of the triazoline cycle (Figure 6).In conclusion, it is worth noting that the 1,3-dipolar cycloaddition of nitrile imines to compounds 2 containing an exocyclic bond C=N resulted in the formation of a heterocyclic product 5 with the jointed imidazolidine and 1,2,4-triazoline fragments.The yields of the products 5 were generally good to excellent, except for those obtained from imines 2f and 2g, whose substituents had pronounced electron-donating or electron-acceptor properties.The formation of complex mixtures comprising the products of the addition of 1 or 2 nitrile imine molecules was observed for the sulfur-containing dipolarophiles 2h-j.The structures of compounds 5c, 5g, and 5i were confirmed by X-ray diffraction analysis.In the reactions of nitrile imine formed from hydrazonoyl halide 3e and dipolarophiles 2b and 2d containing ortho-substituents in arylimino fragments, products 5d and 5e were obtained as a mixture of atropoisomers.Trying to obtain imine from non-symmetrically substituted imidazoletrione 1b, we found that both neighboring C=O groups may undergo the reaction, thus giving a mixture of products (Scheme 8).In contrast to the chemoselective aza-Wittig reaction of the carbonyl group at N-Alkyl claimed in [17], in our case, the major product contained an imino group in a position adjacent to N-Ph.The isomeric products 2k and 2l had almost identical chromatographic properties and were indistinguishable for TLC analysis at CHCl 3 elution (this eluent was used in [17]).But the difference between 2k and 2l retention factors became noticeable in less polar systems (for example, hexane-EtOAc or hexane-DCM) and was maximum in the hexane-Et 2 O system that we used for separation (see Section 3.3).Since X-ray diffraction analysis of 2k and 2l turned out to be unrealizable, a structural organization of the imines was proposed based on the structures of the spiro compounds 5m and 5n obtained by their reaction with hydrazonoyl chloride 3b.The aza-Wittig reaction chemoselectivity observed for substrate 1b can be attributed to partial delocalization of the lone pair of electrons on nitrogen by an aromatic fragment, which enhanced the carbonyl activity of the C 5 atom nearby.The ratio of the isolated products (2k:2l ≈ 1:3) corresponded with the ratio of isomers estimated by the 1 H NMR spectrum of the aliquot taken from the reaction mixtures before separation.
In conclusion, it is worth noting that the 1,3-dipolar cycloaddition of nitrile imines to compounds 2 containing an exocyclic bond C=N resulted in the formation of a heterocyclic product 5 with the jointed imidazolidine and 1,2,4-triazoline fragments.The yields of the products 5 were generally good to excellent, except for those obtained from imines 2f and 2g, whose substituents had pronounced electron-donating or electron-acceptor properties.The formation of complex mixtures comprising the products of the addition of 1 or 2 nitrile imine molecules was observed for the sulfur-containing dipolarophiles 2h-j.The structures of compounds 5c, 5g, and 5i were confirmed by X-ray diffraction analysis.In the reactions of nitrile imine formed from hydrazonoyl halide 3e and dipolarophiles 2b and 2d containing ortho-substituents in arylimino fragments, products 5d and 5e were obtained as a mixture of atropoisomers.

Synthesis and 1,3-Dipolar Cycloaddition Reactions of the Imines 2k и 2l Obtained from Unsimmetrical Ureas
Trying to obtain imine from non-symmetrically substituted imidazoletrione 1b, we found that both neighboring C=O groups may undergo the reaction, thus giving a mixture of products (Scheme 8).In contrast to the chemoselective aza-Wittig reaction of the carbonyl group at N-Alkyl claimed in [17], in our case, the major product contained an imino group in a position adjacent to N-Ph.The isomeric products 2k and 2l had almost identical chromatographic properties and were indistinguishable for TLC analysis at CHCl3 elution (this eluent was used in [17]).But the difference between 2k and 2l retention factors became noticeable in less polar systems (for example, hexane-EtOAc or hexane-DCM) and was maximum in the hexane-Et2O system that we used for separation (see Section 3.3).Since X-ray diffraction analysis of 2k and 2l turned out to be unrealizable, a structural organization of the imines was proposed based on the structures of the spiro compounds 5m and 5n obtained by their reaction with hydrazonoyl chloride 3b.The aza-Wittig reaction chemoselectivity observed for substrate 1b can be attributed to partial delocalization of the lone pair of electrons on nitrogen by an aromatic fragment, which enhanced the carbonyl activity of the C 5 atom nearby.The ratio of the isolated products (2k:2l ≈ 1:3) corresponded with the ratio of isomers estimated by the 1 H NMR spectrum of the aliquot taken from the reaction mixtures before separation.Compounds 2k and 2l were further reacted with hydrazonoyl chloride 3b to form corresponding products 5m and 5n (triethylamine was added dropwise), the structure of which was established on the basis of NMR spectra data.In the 1 H NMR spectrum of the compound 5m, the signals of the CH 2 -group protons at the nitrogen atom of the imidazolidine cycle are presented as two doublets at 4.32 and 4.10 ppm with a coupling constant value of 17.6 Hz (see Supplemental Information), which may be due to the difference in the local surroundings of these protons, which are located near the spiro connection.The aliphatic region of the compound 5n 1 H NMR spectrum was almost identical to the original imine 2l, which indicates the remoteness of this substituent from other fragments of the molecule.
The activity of compounds 2k and 2l in 1,3-dipolar cycloaddition reactions may be generalized as follows: On the one hand, at the identical preparative procedures, the yield of 5n was significantly higher than that obtained in the reaction of the same hydrazonoyl chloride (3b) and imine containing only aromatic substituents (Yield drop (5h) = 50% vs. 76% for 5n).On the other hand, the compound 5m was obtained with a low yield of 15%.At the same time, we observed a very low conversion of imine 2k, which was isolated unchanged at the end of the reaction.
The replacement of phenyl by the -CH 2 COOEt group in imine 2c led to an increase in the nitrogen atom N 3 lone electron pair delocalization between neighboring carbon atoms C 2 and C 4 in both compounds (2k and 2l).Consequently, if the fragment C=N is located at C 4 (imine 2k), the electron density on this atom increases significantly more than in compound 2l, where the arylimino group is located at atom C 5 (due to two factors: first, this atom is more distant from N 3 , and second, N 3 turns out to be surrounded by two C=O groups, delocalizing its electron density).This small increase in the electron density of the C=N bond could contribute to the optimal overlap between the MO imine and the dipole, thereby dramatically raising the yield of the cycloaddition product.

Biological Evaluation of Selected Spiro-Compounds
The cytotoxicity of some spiro derivatives of both structural types 4 and 5 was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test in the human colorectal carcinoma cell line HCT116 (Table 6).Overall, the selected compounds demonstrated moderate cytotoxic activity with IC 50 values in the range of ~25-45 µM.Compounds 2k and 2l were further reacted with hydrazonoyl chloride 3b to form corresponding products 5m and 5n (triethylamine was added dropwise), the structure of which was established on the basis of NMR spectra data.In the 1 H NMR spectrum of the compound 5m, the signals of the CH2-group protons at the nitrogen atom of the imidazolidine cycle are presented as two doublets at 4.32 and 4.10 ppm with a coupling constant value of 17.6 Hz (see Supplemental Information), which may be due to the difference in the local surroundings of these protons, which are located near the spiro connection.The aliphatic region of the compound 5n 1 H NMR spectrum was almost identical to the original imine 2l, which indicates the remoteness of this substituent from other fragments of the molecule.
The activity of compounds 2k and 2l in 1,3-dipolar cycloaddition reactions may be generalized as follows: On the one hand, at the identical preparative procedures, the yield of 5n was significantly higher than that obtained in the reaction of the same hydrazonoyl chloride (3b) and imine containing only aromatic substituents (Yielddrop (5h) = 50% vs. 76% for 5n).On the other hand, the compound 5m was obtained with a low yield of 15%.At the same time, we observed a very low conversion of imine 2k, which was isolated unchanged at the end of the reaction.
The replacement of phenyl by the -CH2COOEt group in imine 2c led to an increase in the nitrogen atom N 3 lone electron pair delocalization between neighboring carbon atoms C 2 and C 4 in both compounds (2k and 2l).Consequently, if the fragment C=N is located at C 4 (imine 2k), the electron density on this atom increases significantly more than in compound 2l, where the arylimino group is located at atom C 5 (due to two factors: first, this atom is more distant from N 3 , and second, N 3 turns out to be surrounded by two C=O groups, delocalizing its electron density).This small increase in the electron density of the C=N bond could contribute to the optimal overlap between the MO imine and the dipole, thereby dramatically raising the yield of the cycloaddition product.

Biological Evaluation of Selected Spiro-Compounds
The cytotoxicity of some spiro derivatives of both structural types 4 and 5 was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test in the human colorectal carcinoma cell line HCT116 (Table 6).Overall, the selected compounds demonstrated moderate cytotoxic activity with IC50 values in the range of ~25-45 µM.Compounds 2k and 2l were further reacted with hydrazonoyl chloride 3b to form corresponding products 5m and 5n (triethylamine was added dropwise), the structure of which was established on the basis of NMR spectra data.In the 1 H NMR spectrum of the compound 5m, the signals of the CH2-group protons at the nitrogen atom of the imidazolidine cycle are presented as two doublets at 4.32 and 4.10 ppm with a coupling constant value of 17.6 Hz (see Supplemental Information), which may be due to the difference in the local surroundings of these protons, which are located near the spiro connection.The aliphatic region of the compound 5n 1 H NMR spectrum was almost identical to the original imine 2l, which indicates the remoteness of this substituent from other fragments of the molecule.
The activity of compounds 2k and 2l in 1,3-dipolar cycloaddition reactions may be generalized as follows: On the one hand, at the identical preparative procedures, the yield of 5n was significantly higher than that obtained in the reaction of the same hydrazonoyl chloride (3b) and imine containing only aromatic substituents (Yielddrop (5h) = 50% vs. 76% for 5n).On the other hand, the compound 5m was obtained with a low yield of 15%.At the same time, we observed a very low conversion of imine 2k, which was isolated unchanged at the end of the reaction.
The replacement of phenyl by the -CH2COOEt group in imine 2c led to an increase in the nitrogen atom N 3 lone electron pair delocalization between neighboring carbon atoms C 2 and C 4 in both compounds (2k and 2l).Consequently, if the fragment C=N is located at C 4 (imine 2k), the electron density on this atom increases significantly more than in compound 2l, where the arylimino group is located at atom C 5 (due to two factors: first, this atom is more distant from N 3 , and second, N 3 turns out to be surrounded by two C=O groups, delocalizing its electron density).This small increase in the electron density of the C=N bond could contribute to the optimal overlap between the MO imine and the dipole, thereby dramatically raising the yield of the cycloaddition product.

Biological Evaluation of Selected Spiro-Compounds
The cytotoxicity of some spiro derivatives of both structural types 4 and 5 was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test in the human colorectal carcinoma cell line HCT116 (Table 6).Overall, the selected compounds demonstrated moderate cytotoxic activity with IC50 values in the range of ~25-45 µM.Compounds 2k and 2l were further reacted with hydrazonoyl chloride 3b to form corresponding products 5m and 5n (triethylamine was added dropwise), the structure of which was established on the basis of NMR spectra data.In the 1 H NMR spectrum of the compound 5m, the signals of the CH2-group protons at the nitrogen atom of the imidazolidine cycle are presented as two doublets at 4.32 and 4.10 ppm with a coupling constant value of 17.6 Hz (see Supplemental Information), which may be due to the difference in the local surroundings of these protons, which are located near the spiro connection.The aliphatic region of the compound 5n 1 H NMR spectrum was almost identical to the original imine 2l, which indicates the remoteness of this substituent from other fragments of the molecule.
The activity of compounds 2k and 2l in 1,3-dipolar cycloaddition reactions may be generalized as follows: On the one hand, at the identical preparative procedures, the yield of 5n was significantly higher than that obtained in the reaction of the same hydrazonoyl chloride (3b) and imine containing only aromatic substituents (Yielddrop (5h) = 50% vs. 76% for 5n).On the other hand, the compound 5m was obtained with a low yield of 15%.At the same time, we observed a very low conversion of imine 2k, which was isolated unchanged at the end of the reaction.
The replacement of phenyl by the -CH2COOEt group in imine 2c led to an increase in the nitrogen atom N 3 lone electron pair delocalization between neighboring carbon atoms C 2 and C 4 in both compounds (2k and 2l).Consequently, if the fragment C=N is located at C 4 (imine 2k), the electron density on this atom increases significantly more than in compound 2l, where the arylimino group is located at atom C 5 (due to two factors: first, this atom is more distant from N 3 , and second, N 3 turns out to be surrounded by two C=O groups, delocalizing its electron density).This small increase in the electron density of the C=N bond could contribute to the optimal overlap between the MO imine and the dipole, thereby dramatically raising the yield of the cycloaddition product.

Biological Evaluation of Selected Spiro-Compounds
The cytotoxicity of some spiro derivatives of both structural types 4 and 5 was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test in the human colorectal carcinoma cell line HCT116 (Table 6).Overall, the selected compounds demonstrated moderate cytotoxic activity with IC50 values in the range of ~25-45 µM.The obtained cytotoxicity results allow us to draw some preliminary conclusions about the effect of substituents in aromatic fragments on the activity of compounds.Firstly, type 4 compounds with a NO2-group in one of the aromatic fragments showed slightly better results (compounds 4a, 4b, and 4f) compared to 4e.On the other hand, compound 5j, which does not contain NO2-groups, showed lower IC50 values compared to 5c and 5f.When comparing the IC50 values for compounds 4b and 4f, it can be noticed that the combination of a substituted aromatic fragment and a CH2COOEt group at nitrogen atoms is somewhat more preferable than the presence of two phenyls.It can also The obtained cytotoxicity results allow us to draw some preliminary conclusions about the effect of substituents in aromatic fragments on the activity of compounds.Firstly, type 4 compounds with a NO 2 -group in one of the aromatic fragments showed slightly better results (compounds 4a, 4b, and 4f) compared to 4e.On the other hand, compound 5j, which does not contain NO 2 -groups, showed lower IC 50 values compared to 5c and 5f.When comparing the IC 50 values for compounds 4b and 4f, it can be noticed that the combination of a substituted aromatic fragment and a CH 2 COOEt group at nitrogen atoms is somewhat more preferable than the presence of two phenyls.It can also be pointed out that the presence of a halogen atom in the R 1 position of the compound 5c led to a slight increase in cytotoxicity compared to 5f (R 1 = CH 3 ).As can be seen from the IC 50 measurements, the cytotoxicity of the presented compounds belongs to the region of micromolar concentrations, which is a good but insufficient value compared to the known lead compounds [12,30].This result may be explained by the high lipophilicity of compounds containing 3-5 aromatic fragments within a relatively small volume of the molecule.

General Information
The reagents were purchased from commercial sources and used without further purification.All solvents used were purified and dehydrated, as described in [31].Reactions were checked by thin layer chromatography (TLC) analysis using silica plates with a fluorescent indicator (254 nm) and visualized with a UV lamp.
The IR spectra were recorded using a Thermo Nicolet iS5 FT-IR Spectrometer (Thermo Electron Scientific Instruments LLC, Madison, WI, USA) with an ATR module.
Electrospray ionization high-resolution mass spectra were recorded in positive ion mode on a TripleTOF 5600+ quadrupole time-of-flight mass spectrometer (ABSciex, Concord, Vaughan, ON, Canada) equipped with a DuoSpray ion source.The following MS parameters were applied: capillary voltage 5.5 kV; nebulizing and curtain gas pressures-15 and 25 psi; respectively; ion source temperature-ambient; declustering potential 20 V; m/z range 100-1200.Elemental compositions of the detected ions were determined based on accurate masses and isotopic distributions using Formula Finder software (ABSciex, Concord, ON, Canada).The maximum allowed deviation of the experimental molecular mass from the calculated one was 5 ppm.
The X-ray data were collected via the STOE diffractometer Pilatus100K detector (DEC-TRIS AG, Baden, Switzerland), Cu Kα (1.54086Å) radiation, and rotation method mode.STOE X-AREA software was used for cell refinement and data reduction.Data collection and image processing were performed with X-Area 1.67 (STOE & Cie GmbH, Darmstadt, Germany, 2013).Intensity data were scaled with LANA (part of X-Area) in order to minimize differences in intensities of symmetry-equivalent reflections (multi-scan method).CCDC 2310170 JK269 (compound 5i), CCDC 2310171 JK314-2 (compound 5g), and CCDC 2310172MB-13 (compound 5c) contain the supplementary crystallographic data for this paper.The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via the www.ccdc.cam.ac.uk/data_request/cif (accessed on 17 December 2023) website.

Cell Lines and Cytotoxicity Evaluation
The human colon adenocarcinoma cell line (HCT116) was purchased from ATCC (Manassas, VA, USA).Cancer cells were routinely grown in RPMI 1640 culture medium, supplemented with 10% fetal bovine serum, glutamine, and 100 U/mL penicillin.HCT116 cell line was grown in flasks in RPMI 1640 fresh culture medium with supplements at 37 • C and 5% CO 2 .Cells were grown monolayer cultures, and the cells in the exponential growth phase were trypsinized and suspended in RPMI 1640 medium supplemented.

Cytotoxicity
To evaluate the cytotoxicity of compounds in vitro, we placed cells in (4-7) × 10 3 cells/mL concentrations in 96-well culture plates for 24 h.Cells were counted after treatment with Trypan blue solution (0.4%).They were then exposed to different concentrations of compounds in two-fold serial (50-100 µM) dilutions in pre-incubated cells at 37 • C for 72 h.In control wells with untreated cells, only (DMCO + PBS) were added.Cell viability was measured by the standard MTT test [31].The absorbance was measured at 540 nm using a Multiskan™ FC microplate photometer and Skanlt software 6.1 RE for microplate readers, both from Thermo Scientific (Waltham, MA, USA).

Statistical Analysis
In vitro experiments were carried out in triplicate.Graphpad Prism version 9.0 was used to determine the IC50.The data for IC50 are presented as the mean ± standard deviation (SD).

Conclusions
A series of new spiro derivatives containing pyrazoline and imidazoledione fragments were synthesized by 1,3-dipolar cycloaddition of nitrile imines, which were generated in situ from hydrazonoyl chlorides, to the parabanic acid derivatives.It was shown that the cycloaddition reactions proceed regioselectively in all cases, regardless of the presence of electron-donating or electron-withdrawing substituents in the 1,3-dipole molecules.The study of the 1,3-dipolar cycloaddition reaction of nitrile imines by DFT calculation methods showed that 1,3-dipoles act as nucleophiles in the reaction with 3-phenyl-5methylidenehydantoin, and the 1,3-dipolar cycloaddition reaction with normal electronic demands (NED) should have been realized.
The reactions of 2-thioxoimidazolidine-4,5-diones and 5-arylimino-1,3diphenylimidazolidine-2,4,5-triones with nitrile imines were carried out using two alternative synthetic approaches, which differed in the method of adding a base generating a dipole from hydrazonoyl chloride ("classic" dropwise addition method and a diffusion mixing technique).It was found that the diffusion mixing technique typically gives the same or better yield of the target product compared to the addition of amine drop by drop.
The cytotoxicity of some of the obtained spiro derivatives was evaluated using the MTT test on the human colorectal carcinoma cell line HCT116.The tested compounds demonstrated moderate cytotoxic activity with IC 50 values in the range of ~25-45 µM.

Figure 4 .
Figure 4. Structures of the hydrazonoyl chlorides 3a-g used in this study.

Figure 4 .
Figure 4. Structures of the hydrazonoyl chlorides 3a-g used in this study.

Figure 4 .
Figure 4. Structures of the hydrazonoyl chlorides 3a-g used in this study.

Scheme 7 .Scheme 6 .
Scheme 7. The proposed mechanism of 3-phenyl-2-(phenylimino)thiazolidine-4,5-dione 6 and nitrile imine interaction.Based on the obtained results, it can be concluded that 2-thioxoimidazolidine-4,5-diones 1c-f possess the capability to undergo the 1,3-dipolar cycloaddition with nitrile imines obtained in situ from the corresponding hydrazonoyl chlorides 3a-d.As a result, heterocycles 4a-g consisting of spiro-conjugated imidazolidine and 1,2,4-thiadiazoline fragments were formed.The nitrile imine generation can be accomplished through both the "classical" method of adding the base drop by drop and the previously proposed diffusion mixing technique.The use of the latter enabled the

Figure 6 .
Figure 6.Hindered rotation in compounds 5d and 5e is caused by the presence of ortho-substituents in aryl fragments.

Figure 6 .
Figure 6.Hindered rotation in compounds 5d and 5e is caused by the presence of ortho-substituents in aryl fragments.

diones 1c-f and hydrazonoyl chlorides 3a-d.
a Isolated yield; b Yield was not defined.

diones 1c-f and hydrazonoyl chlorides 3a-d.
a Isolated yield; b Yield was not defined.

4 Diffusion Mixing Dropwise Addition Yield a , % Yield a , %
a Isolated yield; b Yield was not defined.

Table 4 .
The influence of reaction conditions on the formation of a by-product.

Table 4 .
The influence of reaction conditions on the formation of a by-product.
a Isolated yield, diffusion mixing technique; b Traces.
a Isolated yield.